This invention relates to protein complexes and use thereof in medical applications. More specifically, it relates to complexes of hemoglobin compounds with therapeutic substances such as drugs, genes etc. which have a therapeutic action on specific parts and/or organs of the body, and means for targeting such complexes to specific body parts and body organs. Also within the scope of the invention are complexes of hemoglobin with diagnostic substances, such as imaging agents.
The use of hemoglobin and modified hemoglobin as a drug delivery means has been proposed previously. Hemoglobin, as a natural component of red blood cells, present and circulating throughout the body in relatively large quantities, has well-established bioacceptability and the potential-to deliver drugs throughout the body.
Thus, Kluger et al., U.S. Pat. No. 5,399,671 describe a hemoglobin compound which has been cross-linked to effect intramolecular stabilization of the tetrameric structure thereof, but which contains a residual functional group on the cross-linker residue to which drugs for delivery can be covalently attached.
Anderson et al., U.S. Pat. No. 5,679,777, describe complexes of hemoglobin compounds and polypeptide drugs, in which the polypeptide drug is bound to a globin chain through a disulfide linkage to a cysteine unit inherent in or genetically engineered into the globin chain.
Haptoglobins (Hp) constitute part of the xcex12-globin family of serum glycoproteins. Haptoglobins are present in mammalian plasma, and constitute about one-quarter of the xcex12-globulin fraction of human plasma. Each individual has one of three phenotypic forms of haptoglobin, of close structural and chemical identity. Haptoglobins are composed of multiple xcex1xcex2 dimers and the phenotypes are conventionally denoted Hp 1-1, Hp 2-1 and Hp 2-2. The xcex2 chains are identical in all haptoglobin phenotypes, but the xcex1 chains vary (xcex11 and xcex12). The amino acid sequences of all chains are known. Hp 1-1 is composed of two xcex11xcex2 dimers and has a molecular weight of about 98 kDa. The structure of Hp 2-1 and Hp 2-2 can be written as follows: (xcex11xcex2)2(xcex12xcex2)n where n=0,1,2, . . . and (xcex12xcex2)m where m=3,4,5, . . . respectively.
Delivery of drugs to a patient suffering from a disease or disorder affecting primarily one body part or one body organ is best accomplished by choosing a delivery method which targets the part or organ in need of treatment with a high degree of specificity. Such a delivery system makes most effective use of the active drug, so as to reduce the necessary dosage level, and reduces side effects of the drug.
One function of haptoglobin is to bind extracellular hemoglobin, arising from red blood cell lysis, to form essentially irreversible haptoglobin-hemoglobin complexes which are recognized by specific receptors on hepatocytes in the liver. In this way, hemoglobin is targeted to the liver for metabolism.
Control and manipulation of genes and gene products are potentially powerful means of treating various diseases and genetic disorders. When specifically introduced into the cells, genes can use the host cell biosynthetic machinery for the expression of the therapeutic biomolecules they encode. For successful gene therapy, one must devise a successful method of in vivo gene delivery. One such technique developed in recent years is receptor-mediated delivery. This has the advantage of high specificity of delivery to the cells which express the targeted receptor.
The specific targeting of low molecular weight therapeutic and diagnostic agents to tissues is enhanced greatly through the use of receptor-mediated delivery. Diagnostic agents such as fluorescent or radiolabeled substances indicate the location and quantity of cells bearing the targeted receptors when such agents are administered as complexes with ligands for those receptors. These complexes are also useful in characterizing the binding and transport properties of receptors on cells in culture. Such information is useful in detection of and/or design of therapy for tissues containing the cells being recognized, either in vitro or in vivo.
It is an object of the present invention to provide a means and composition for specifically targeting hepatocytes or other cells having receptors for hemoglobin-haptoglobin complexes with therapeutically active substances or diagnostic agents.
It is a further object of the present invention to provide a novel complex of a substance selected to exert a beneficial effect on a mammalian patient""s liver, in vivo, and a substance which specifically targets hepatic cells.
The present invention describes haptoglobin-hemoglobin construct-complexes to which hepatocyte-modifying agents are attached. Such haptoglobin-hemoglobin construct-complexes serve as effective hepatocyte-targeting vehicles for the attached agents, for delivery of specific hepatocyte-modifying agents (drugs, diagnostics, imaging compounds, etc) to the liver, and to other cells having the appropriate hemoglobin-haptoglobin receptors.
The expression xe2x80x9cconstruct-complexxe2x80x9d is used herein to refer to the combination of haptoglobin with hemoglobin to which a bioactive, therapeutic or diagnostic agent is attached. The present invention provides construct-complexes composed of a hemoglobin compound, a haptoglobin and a hepatocyte-modifying substance of interest such as a drug, a diagnostic agent or a gene. In one aspect of the present invention, the construct-complex is prepared extracorporeally and then administered to the patient. In another aspect, a complex of hemoglobin-hepatocyte modifying substance is prepared extracorporeally, administered to the patient, and forms the construct-complex of haptoglobin-hemoglobin-hepatocyte modifying substance with haptoglobin which is naturally present in the patient""s serum. In a further aspect, the patient""s haptoglobin level may be supplemented by haptoglobin administration, a known procedure, either before, during or after administration of the hemoglobin-hepatocyte modifying substance-construct-complex. In any case, the construct-complex specifically targets and binds as a ligand to the hepatocyte receptors, owing to the presence of the haptoglobin and hemoglobin portions of the construct-complex.
The construct-complexes of the present invention, formed ex vivo or in vivo, target any cells having receptors for Hb-Hp complexes, and this includes metastases arising from primary hepatoma. It is normally difficult to identify and treat metastases because of the systemic distribution and small size of such cancers. Secondary hepatic metastases, i.e. hepatoma cells outside the liver which have such receptors are targeted by the construct-complexes of the present invention, as well as cells of the liver, and should be regarded as xe2x80x9chepatocytesxe2x80x9d as the term is used herein.
Further, the construct-complexes of the present invention may exert beneficial effects on neighboring cells, if the hepatocyte modifying substance is, for example, a drug which is active towards neighboring cells even if they are not hepatocytes. They may also modulate or initiate the activity of other therapeutic or diagnostic agents delivered by other methods for hepatocyte modification, such as prodrugs, enzymes or genes coding for enzymes and requiring activation to cause an effect. Agents effecting such action resulting in hepatocyte modification or effect on other agents or cells are hepatocyte modifying agents according to this invention.
The construct-complex according to the present invention can be generally represented by the formula:
(Hp)axe2x80x94(Hb)bxe2x80x94(Lcxe2x80x94Ad)e
where
a=1 to about 10;
b=0.5 to about 10;
c=0 to about 10;
d=1 to about 20;
e=1 to about 20;
Hp is haptoglobin as described herein;
Hb is a hemoglobin as described herein;
L is a linker as described herein; and
A is a hepatocyte modifying agent as described herein,
in which the stoichiometry of Hp to Hb in the complex is dictated by the available number of binding sites on the two proteins, but is generally of the order of 1:05 to 1:2.